We have developed a new technique for the immunoassay of clotting factors. The technique is similar to the ELISA methodology in that enzyme-labeled derivatives of antigens and antibodies are used to detect immune complexes. It differs from most ELISA methods in that it utilizes an activator of coagulation (Russell's viper venom factor X activator or RVV-XA) as the labeling enzyme, and a unique clotting assay, entitled enzyme-linked coagulation assay or ELCA, as the detection system. ELCA uses peroxidase-labeled fibrinogen in solution and unlabeled fibrinogen together as a substrate for thrombin which is generated in the clotting cascade; thrombin cleaves both forms to fibrin and consequently peroxidase-fibrin is bound to the solid phase. Used as a detection system for RVV-XA, this assay can measure as little as 50 femtograms/ml of RVV-XA, which in our hands is more than three orders of magnitude lower than the detection limit for peroxidase. We have found that we can label antigens and antibodies with this enzyme and use these conjugates for competitive immunoassays with high sensitivity. By combination of the specificity of monoclonal antibodies for factors of interest (II, VII, IX, X and protein C) with this sensitive detection system, we will assay three important parameters of interest in thrombosis and hemostasis, i.e., activated factor X, clotting factor antigen levels, and complexes of factor Xa with antithrombin III. These immunoassays, in conjunction with the sensitive functional detection of clotting factors by ELCA, will provide a complete system for the detection of components of interest in hemostasis and thrombosis.